Failure of Cyclosporin A to RescuePeripheral Nerve Allografts in AcuteRejection
Delphine L. Chen, MDSusan E. Mackinnon, MDJohn N. Jensen, MDDaniel A. Hunter, R.T.Aaron G. Grand, MD
Prevention and control of graft rejection remain essential in theinvestigation of peripheral nerve allotransplantation. Althoughuse of cyclosporin A (CsA) has been shown to suppress success-fully the rejection of nerve allografts, limited information existsconcerning use of this drug to arrest rejection in progress, andthereby effect salvage of these grafts. The aim of this study wasto determine the efficacy of CsA in the treatment of ongoingacute rejection of peripheral nerve allografts. Buffalo ratsreceived posterior tibial nerve grafts from either Lewis orBuffalo donor animals and were divided into five groups: group1 received isografts and no CsA treatment (n � 8), group 2received allografts with continuous CsA therapy (n � 10),group 3 received allografts with no treatment (n � 7), group 4received allografts with initiation of CsA therapy delayed until3 weeks after the procedure (n � 11), and group 5 receivedallografts with an interrupted course of CsA (n � 15). All graftswere harvested at 10 weeks. Histomorphometric analysis dem-onstrated comparable nerve regeneration in groups 1 and 2 andgood regeneration in group 3 animals, despite cellular infiltratesuggestive of rejection. At 3 weeks after surgery, group 4animals showed early rejection and significantly less neurore-generation than positive controls at 10 weeks after delayedinitiation of CsA therapy. Finally, group 5 animals showed earlyregeneration at 3 weeks but significantly lesser regeneration by10 weeks after interruption of therapy. In this experimentalprotocol, CsA was ineffective in rescuing histologically provenrejection in progress.
Chen DL, Mackinnon SE, Jensen JN, Hunter DA, Grand AG. Failure ofcyclosporin A to rescue peripheral nerve allografts in acute rejection. AnnPlast Surg 2002;49:660–667
From the Division of Plastic and Reconstructive Surgery, WashingtonUniversity School of Medicine, St. Louis, Missouri.
Received Jan 29, 2002. Accepted for publication Apr 18, 2002.
Address correspondence to Susan E. Mackinnon, MD, Division of Plasticand Reconstructive Surgery, Suite 17424, One Barnes-Jewish HospitalPlaza, St. Louis, MO 63110.
The use of cyclosporin A (CsA) in promoting theacceptance of peripheral nerve allografts hasbeen studied extensively. Numerous studies haveshown that reinnervation can occur throughnerve allografts if continuous immunosuppres-
sion with CsA is used and that functional recov-ery in these cases approximates that of isografts.1–6
Other studies have suggested that only temporaryimmunosuppression is required, presumably be-cause axons can survive a rejection reaction di-rected at the nerve sheath as soon as the axons havereached their target organ.2,4,6 However, althoughCsA has been used as rescue therapy for solid organtransplants,7–10 it is not known whether CsA iseffective in halting and rescuing acute rejection ofnerve allografts after rejection has begun. The pur-pose of this study was to examine the effectivenessof CsA in interrupting acute rejection of peripheralnerve allografts.
Materials and Methods
Animal Model and Experimental DesignAdult male inbred Buffalo (RT1b) and Lewis(RT11) rats, weighing 190 to 295 g, were chosenfor this study because they differ at one majorhistocompatibility locus, briskly reject reciprocalnerves without immunosuppression, and can beassessed for rejection using a mixed lymphocytereaction assay. All animals were housed in acentral animal care facility with free access towater and standard rat chow. All housing, care,and operations were in accordance with the Na-tional Institutes of Health Guide for the Care andUse of Laboratory Animals. The study protocolwas approved by this institution’s animal studiescommittee.
Rats were randomized to five groups, withBuffalo rats serving as recipients of posteriortibial nerve grafts in all groups. Group 1 (n � 8)served as an untreated isograft control group inwhich the recipient Buffalo rats received Buffalodonor nerves without any CsA treatment. Group
2 (n � 10) served as a treated allograft controlgroup in which Buffalo rats receiving Lewis allo-grafts received CsA at a dose of 5mg/kg per day,11
from 2 days before surgery until the end of theexperiment. This dosing regimen has been estab-lished previously as providing reliable, stableimmunosuppression to prevent graft rejection inthis model.12 Group 3 (n � 7) served as theuntreated allograft control group. Group 4 (n �
11) rats received allografts and were not treatedwith CsA until 3 weeks after surgery. Group 5 (n� 15) rats received Lewis allografts and weretreated with an interrupted course of CsA. Theseanimals received CsA from 2 days before surgeryuntil 3 weeks after surgery, at which time CsAwas stopped. Cyclosporin A treatment was thenresumed at 6 weeks after surgery until the end ofthe experiment. Figure 1 summarizes the timecourse of CsA administration for the five groups.Cyclosporin A was started before surgery toachieve therapeutic serum levels.
Three randomly selected animals each fromgroups 4 and 5 were killed at 3 weeks for histo-logic and in vitro immunologic studies, to con-firm rejection in group 4 and acceptance of thegraft in group 5. Another three animals fromgroup 5 were selected randomly for sacrifice at 6weeks, also for histologic and in vitro immuno-logic assays to confirm rejection. At 10 weeks, allremaining animals were given a lethal injectionof pentobarbital sodium (DelMarva Laboratories,Midlothian, VA), and the nerves harvested forhistologic analysis.
Surgical TechniqueAnesthesia was induced with an intramuscularinjection of ketamine hydrochloride (FortDodge Animal Health, Fort Dodge, IA) at 30
mg/kg and medetomidine hydrochloride (PfizerAnimal Health, Exton, PA) at 0.2 mg/kg. Surgi-cal procedures were performed aseptically withan operating microscope (Wild-Heerbrugg;Wild-Leitz Canada, Willowdale, Ontario, Can-ada). Exposure of the posterior tibial nerve wasachieved through a lateral biceps femoris mus-cle-splitting incision. An external neurolysistechnique was used to isolate the posteriortibial nerve, which was then transected. A1.8-cm nerve graft was placed at the point oftransection, and a tension-free, end-to-endepineurial repair was performed using micro-surgical techniques and a 10-0 nylon suture.Muscle and skin were closed with 4-0 Vicryland 5-0 nylon interrupted sutures (Ethicon,Somerville, NJ), respectively.
Immunosuppressive RegimenOral CsA (SandImmune; Sandoz, East Hanover,NJ) was diluted to 15 mg/mL with a mixture of80% ethanol (Quantum Chemical, Tuscola, IL)and 20% Tween 80 (Fisher Chemical, FairLawn, NJ) and administered once daily byintraperitoneal injection beginning 2 days be-fore surgery. The amount of CsA administeredwas adjusted weekly according to weight tomaintain a 5 mg/kg per day dosage for eachanimal. Each rat was monitored by daily obser-vation for loose stools, dehydration, and weightloss.
Mixed Lymphocyte ReactionThree randomly selected animals from groups 4and 5 were killed at either 3 weeks or 6 weeks toassess acceptance or rejection of the nerve graft.Splenectomy was performed, and 2.5 � 105
splenocytes were harvested from experimental
Fig 1. Time course ofcyclosporin A administrationover the 10-week period ofthe experiment.
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animals and co-cultured with 2.5 � 105 gamma-irradiated Lewis or Buffalo splenocytes (total vol-ume, 200 �L) in a 96-well flat-bottomedmicrotiter plate (Costar, Cambridge, MA).13 Sim-ilarly, splenocytes were harvested from naïveanimals and co-cultured in an identical fashionto function as controls. Complete Iscove’s modi-fied Dulbecco’s tissue culture medium was sup-plemented with 1% L-glutamine, 100,000 U/Lpenicillin-streptomycin, 10 mmol/L HEPES, 1%sodium pyruvate, 1% nonessential amino acids,1 � 10�5 M mercaptoethanol, and 10% fetal calfserum (Hyclone Laboratories, Logan, UT). Cellswere incubated for 5 days at 37°C in 5% CO2 withhumidification. Two �Ci of [3H]-thymidine werethen added to each well 16 hours before harvest-ing, and radiolabel incorporation was determinedin a liquid scintillation counter (1,214 Rackbeta;LKB, Turku, Finland).
Histomorphometric EvaluationThe nerve graft and the host nerve proximal anddistal to the graft were harvested en bloc andfixed in an immersion of cold 3% glutaraldehydesolution in 0.1 mol/L phosphate buffer (Poly-sciences, Warrington, PA), with pH adjusted to7.2. The segments were then postfixed with 1%osmium tetroxide and embedded in Araldite 502(Polysciences). One-micrometer thin sectionsfrom the proximal and distal host nerve as well asthe nerve graft were taken and stained withtoluidine blue (Polysciences) for examination un-der light microscopy. Cross-sections 5 mm distalto the nerve graft were used to calculate totalfascicular area and total fiber numbers. The mi-croscopic image was digitized and displayed on amonitor calibrated at 0.125 �m/pixel. Computeranalysis of the digitized slide was based on grayand white scales. A minimum of 500 myelinatedfibers or six randomly selected fields per nervewere evaluated for myelin width, axon width,and fiber diameter at 1,000� magnification withan automated digital image analysis systemlinked to morphometry software (Leco Instru-ments, St. Joseph, MI). These primary measure-ments were used to calculate nerve fiber density(fibers/mm2), total number of myelinated fibers,percentage of neural tissue (100 � neural area/fascicular area), percentage of myelin debris (100
� area of debris/fascicular area), and nerve fiberwidth.
Statistical AnalysisThe mean � standard deviation was used topresent all data in this study. A one-tailed anal-ysis of variance was used to determine differ-ences between groups for histomorphometricanalysis. If significant, a least-significance testwas performed to compare groups. All calcula-tions were performed with statistical software forpersonal computers. Histomorphometric calcula-tions were performed using Statistica (StatSoft,Tulsa, OK). Statistical significance was estab-lished at p � 0.05.
Results
General ObservationsAll rats were healthy and gained weight, exceptfor two rats in each of groups 2, 4, and 5.Peritonitis developed in these rats after intraperi-toneal injection and they died by 5 weeks aftersurgery. These six animals were excluded fromanalysis and replaced.
Mixed Lymphocyte ReactionThree rats from group 4 were killed 3 weeks aftersurgery, having received no CsA therapy. Theseanimals had elevated reactivity to Lewis rat cellson the mixed lymphocyte reaction assay with amean stimulation index of 5.9, suggestive ofacute rejection. Conversely, 3 weeks after sur-gery, the animals from group 5 had receivedcontinuous CsA and showed low mixed lympho-cyte reaction reactivity. These animals had amean stimulation index of 1.1, indicative of alack of immunologic rejection. At 6 weeks aftersurgery, mixed lymphocyte reaction assays fromanimals in group 5 showed moderate reactivity toLewis antigen with a mean stimulation index of2.1, indicating a mild or moderate immune re-sponse to the allograft.
Histomorphometric EvaluationControl groups included all animals in groups 1,2, and 3. In addition, animals in groups 4 and 5killed 3 weeks after surgery in groups 4 and 5served as negative and positive controls for his-tologic evidence for rejection. All animals in
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groups 1, 2, and 3 demonstrated evidence ofeffective nerve regeneration at 10 weeks aftersurgery. There was no statistically significanthistomorphometric difference between any pa-rameters in these groups except in percent nervewhen comparing the isograft and untreated allo-graft groups (p � 0.03). However, histologic ex-amination of group 3 nerves demonstrated amoderate or large degree of cellular infiltrate,indicative of inflammation and rejection. Thisinfiltrate was not noted in the positive controlgroups (groups 1 or 2). Figure 2 shows represen-tative histologic analysis for all groups at 10weeks after surgery. Figure 3 shows the histomor-phometric data for all groups at 10 weeks aftersurgery.
At the 3-week time point, histologic examina-tion confirmed rejection in the three animalskilled from group 4, the delayed-treatment group.No regenerating axons could be identified in thedistal portion of these nerves. In the animals fromgroup 5 (interrupted treatment), the total numberof nerve fibers was 116 � 159, the percentage ofneural tissue was 0.16% � 0.23%, fiber densitywas 218 � 308 fibers/mm2, percentage of myelindebris was 16% � 4%, and fiber width was 1.08� 1.48 �m. These differences were not statisti-cally significant, indicating a lack of significantdistal nerve regeneration at 3 weeks.
Histologic examination of animals from group 5(interrupted treatment) at 6 weeks after surgeryconfirmed rejection in all animals. After 3 weeksof treatment with CsA followed by 3 weeks with-out CsA, the total number of nerve fibers was 611� 757, percentage of neural tissue was 1.55% �
2.02%, fiber density was 2217 � 2894 fibers/mm2, percentage of myelin debris was 9% � 1%,and fiber width was 1.67 � 1.45 �m. Althoughthere were no time-matched controls for theseanimals, all these numbers were statistically sig-nificantly lower than those for groups 1, 2, or 3 (p� 0.025).
Animals in group 4 were allowed to reject for 3weeks before the initiation of CsA therapy. At 10weeks, these nerves demonstrated evidence ofcellular infiltrate and rejection on histologic ex-amination. Histomorphometric analysis demon-strated statistically significantly lower percentageof neural tissue than either of the positive controlgroup (groups 1 or 2; p � 0.0007). Similarly, there
was a significantly lower nerve density thaneither positive control group (p � 0.02). Therewere no statistically significant differences rela-tive to nerves from animals in the negative con-trol group (group 3).
Group 5, which received an interrupted courseof CsA, had significantly lower total numbers ofnerve fibers, percentage of neural tissue, andnerve density than the positive control groups (p� 0.007, p �0.0002, and p � 0.0004, respective-ly). These nerves also had a significantly lowerpercentage of neural tissue and nerve densitythan the untreated allograft control group (group3; p � 0.04 and p � 0.03, respectively).
There were no statistically significant differ-ences in fiber width between any groups.
Discussion
Unlike solid organ transplantation, indefiniteimmunosuppression in peripheral nerve allo-transplantation is not needed. Because the in-flammatory reaction is directed at the antigenicSchwann’s cells within the allograft, such arejection response can result in the disruptionof regenerating axons and can render the allo-graft incapable of supporting nerve regenera-tion.14 However, as soon as these regeneratinghost axons have traversed the allograft andreached their host motor or sensory targets,systemic immunosuppression can be with-drawn.4,15–17 After this time, a limited rejectionresponse can be tolerated with survival of ex-isting axons and donor support cells.18
Many studies have proven the efficacy of CsAin preventing this type of rejection,2,4,6,15–18 butno study has evaluated the ability of CsA torescue nerve allografts undergoing rejection inprogress. In theory, if the rejection response canbe interrupted before the Schwann’s cells,Schwann’s cell basal lamina, and endotheliumare completely destroyed, then it may be possibleto effect this type of rescue.
In solid-organ transplantation, investigatorshave examined the use of CsA as rescue therapyfor rejection in progress.9,10,19 However, thesestudies did so in protocols that either did notinclude CsA in the primary immunosuppressiveregimen7,8 or attempted to alter the route of CsA
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administration to increase local drug levels, as inthe use of aerosolized CsA therapy in acute rejec-tion of lung transplants.9,10 Although direct ap-plication of CsA to the graft at the time oftransplantation may result in more effective im-munosuppression initially, deeply embeddednerve allografts would not be amenable to directapplication of CsA as rescue therapy.
Despite these limitations, CsA retains interestas a potential agent for the treatment of rejection-in-progress of peripheral nerve allografts. Its rel-
atively low cost, stable dosing regimen, and longexperience in clinical use demonstrates that ithas an application in nerve allografts.20 The im-munosuppressive requirement for the peripheralnerve allograft, although certainly present, ap-pears to be less stringent in the degree of suppres-sion and the period of time required. Thesefeatures make CsA an attractive candidate for thisrole, despite its relatively poor success in therescue of solid organ allografts.
The experimental design included three control
Fig 2. Light microscopy of nerve segments immediatelydistal to graft. Toluidine blue, scale bar � 10 �m. (A)Untreated isograft. Axons are clearly visible as blackannulae. Nerve fiber density is greater than in panels (C),(D), and (E). (B) Treated allografts. Axons are clearlyvisible with myelination evident. (C) Untreated allograft.Axons are less distinct, myelination decreased, and moredebris evident. (D) Delayed treatment. Axons are lessdistinct, with decreased myelination and nerve fiberdensity. (E) Interrupted treatment. Structural damage isevident as in panels (C) and (D).
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groups and two experimental groups. Three weekswithout immunosuppression has been shown to beadequate to allow nearly complete rejection ofnerve allografts based on histologic studies on thesequence of events in nerve allograft rejection.21–23
Thus, the 3-week periods without CsA treatment inthe delayed-treatment and interrupted-treatmentgroups allowed histologic and immunologic docu-mentation of rejection to be made. The delayed-treatment group was designed to address whethercomplete rejection could be reversed with CsA, andthe interrupted-treatment group was designed todetermine whether a nerve could be rescued in aclinically relevant situation where immunosup-pressive therapy was first given and then withheld.
Histomorphometric analysis of nerves from thecontrol groups showed nerve regeneration pa-rameters consistent with prior experiments in ourlaboratory. The untreated allograft group showedhistologic evidence of rejection, correlating withpoor functional recovery by walking track analy-sis (data not shown). This histomorphometric
evidence of nerve regeneration through a reject-ing allograft demonstrates the remarkable neuro-regenerative capabilities of the rat.
The treated allograft and untreated isograft con-trols both showed acceptance of the graft, corre-lating with reasonable functional recovery.Histologic analysis of the delayed-treatment andinterrupted-treatment groups showed that, after 3weeks without CsA treatment, total number ofnerve fibers in both groups was low, indicatingthat the allografts could not be salvaged in eithergroup using the maintenance dose of 5 mg/kg perday of CsA. This is most likely because, after thisperiod of time, the rejection process had pro-gressed to the point where any remainingSchwann’s cells and Schwann’s cell basal laminawere incapable of supporting regenerating axons.
Of note, there may be an inverse relationshipbetween myelin debris and percentage neuraltissue. Because the measurement of myelin de-bris incorporates both the infiltrating leukocytesand their inflammatory effects, immunosuppres-
Fig 3. Bar graphs for allhistomorphometric parameters forall five groups 10 weeks aftersurgery.
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sion logically would result in a decrease in mye-lin debris. Additionally, in some studies, CsA hasbeen noted to have a neuroregenerative effectindependent of its immunosuppressive effect.2,18
These effects of CsA may thus be synergistic,improving nerve regeneration and reducing thedegree of Wallerian degeneration and myelindebris.
In this study, we found that we could notrescue nerves that had nearly completed therejection process before an immunosuppressiveregimen was started. It is possible that initiatingCsA at an earlier stage in the rejection processmay salvage a nerve allograft.
However, rejection is an insidious processand, unlike many solid-organ allografts, whichare immediately functional, there is no loss offunction to herald nerve allograft rejection. Noserum analysis is available to assess nerverejection, and biopsy of the allograft itself is notfeasible because of its size, location, and func-tion as a conduit for regenerating axons. Nerverejection is assessed clinically by indurationand erythema over the graft and a nonprogress-ing Tinel’s sign. In our clinical practice, weroutinely place a short segment of allograft in asubcutaneous location in a distant location forclinical monitoring or histologic assessment. Itis conceivable that rejection may proceed to thepoint of allograft destruction before becomingclinically detectable. Further studies to iden-tify methods of early detection of nerve rejec-tion are needed to improve outcomes afternerve allografting. In addition, Tacrolimus hasbeen shown to be effective when used as rescuetherapy in liver,24 renal,25 heart,26 and lung27
transplant recipients and has shown promise inthe rescue of nerve allografts.28
This study clearly showed that untreated nerveallografts elicit rejection responses that result indestruction of the transplanted tissue within 3weeks. Administration of CsA after this time wasunable to rescue these allografts to allow fornerve regeneration. Although immunosuppres-sion is only required until the regenerating hostaxons have traversed the nerve allograft, theinability to detect rejection in such a scenariomakes it difficult to initiate rescue therapypromptly. Thus, significant damage to the allo-graft may occur, making it difficult or impossible
to effect salvage of the graft by institution oftherapeutic doses of CsA.
Supported by the National Institutes of Health, Bethesda, Maryland (grantno. NIH RO1 NS33406-04).
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